Levy1 project protocol

Bone microarchitecture in 31 emerging lines of the Collaborative Cross (pre-CC)   (2015)

Levy R, Mott RF, Iraqi FA, Gabet Y

See also: Levy1 animal documentation


Project protocol - Contents

Workflow and sampling

Step
Procedure
Equipment
Data collected
1
Mice are euthanized Isoflurane inhalation
-
2 Left femurs are harvested and fixed - -
3 Femurs are analyzed via µCT µCT system Parameters determined in the metaphyseal trabecular bone

Equipment and supplies

  • µCT System (µCT 50, Scanco Medical AG, Switzerland)

Reagents and solutions

  • 5% isoflurane
  • 4% paraformaldehyde
  • 70% ethanol

Procedure: Femur preparation and µCT evaluation

    1. Mice are intraperitoneally euthanized with cervical dislocation performed approximately one min after breathing stops owing to 5% isoflurane inhalation.
    2. Left femurs are harvested and fixed for 24h in 4% paraformaldehyde solution, and then stored in 70% ethanol.
    3. Whole femurs are examined by a µCT system as previously described (Hiram-Bab et al., 2015).
    4. Scans are performed at a 10 µm resolution in all three spatial dimensions.
    5. Mineralized tissues are differentially segmented by a global thresholding procedure as described by Ruegsegger et al, 1996.
    6. All morphometric parameters are determined by a direct 3D approach as described by Hildebrand et al., 1999.
    7. Parameters analyzed are determined in the metaphyseal trabecular bone.

    References

    Hildebrand T, Laib A, Muller R, Dequeker J, Ruegsegger P. Direct three-dimensional morphometric analysis of human cancellous bone: microstructural data from spine, femur, iliac crest, and calcaneus. J Bone Miner Res. 1999. 14:1167-74.

    Hiram-Bab S, Liron T, Deshet-Unger N, Mittleman M, Gassmann M, Rauner M, et al., Erythropoietin directly stimulates osteoclast precursors and induces bone loss. FASEB J. 2015. 29:1890-900.

    Ruegsegger P, Koller B, Muller R. A microtomographic system for the nondestructive evaluation of bone architecture. Calcif Tissue Int. 1996. 58:24-9.

Data collected by investigator

  • Femur length
  • Trabecular bone volume fraction
  • Trabecular thickness
  • Trabecular number
  • Trabecular connectivity density